EP4275701A1 - Régime de conditionnement pour transplantation cellulaire - Google Patents

Régime de conditionnement pour transplantation cellulaire Download PDF

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Publication number
EP4275701A1
EP4275701A1 EP22173383.5A EP22173383A EP4275701A1 EP 4275701 A1 EP4275701 A1 EP 4275701A1 EP 22173383 A EP22173383 A EP 22173383A EP 4275701 A1 EP4275701 A1 EP 4275701A1
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igg
cell
cells
subject
alemtuzumab
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Hansa Biopharma AB
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Hansa Biopharma AB
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Priority to EP22173383.5A priority Critical patent/EP4275701A1/fr
Priority to PCT/EP2023/062850 priority patent/WO2023218078A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)

Definitions

  • the present invention relates to a conditioning regimen for the transplant of a cell, optionally hematopoietic stem / progenitor cells (HSPC), to a subject.
  • HSPC hematopoietic stem / progenitor cells
  • the invention also relates to methods for the prevention or treatment of a disease or condition in a subject by administration of a cell transplant, wherein said administration comprises the conditioning regimen of the invention.
  • HVG Vigorous host versus graft reactions
  • GVHD graft versus host disease
  • radiation may be used to deplete some or all of the existing bone marrow cells in the recipient, creating space for engraftment of the transplanted cells.
  • lymphocyte depleting agents are often used. Using high doses of these agents leads to a desired reduction of GvHD, but at the cost of delayed immune reconstitution or even increased host engraftment failure (because the agents also act on the transplanted cells), thereby enhancing the risk of severe infectious complications and reducing overall survival (OS).
  • OS overall survival
  • Conditioning regimens for the transplant of cells typically include a step of reducing the numbers and/or down-modulating the activity of lymphocytes (preferably T lymphocytes) in the subject prior to transplant. This may be referred to as lymphocyte depletion, or lymphodepletion. It may be achieved by administration of an agent comprising IgG molecules.
  • lymphocyte depletion or lymphodepletion.
  • IgG molecules include rabbit antithymocyte globulin (rATG) or an anti-CD52 monoclonal antibody (e.g. alemtuzumab, CAMPATH).
  • lymphocyte numbers and/or activity Whilst a reduction in lymphocyte numbers and/or activity may be beneficial in terms of improved cell engraftment, it of course also has negative effects, in that the agents responsible may also attack the transplanted cells, and the patient will have a less effective immune system until the effects are reversed, e.g. by recovery of lymphocyte numbers.
  • conditioning regimens may use a lower intensity lymphocyte depletion (e.g. lower / less frequent doses of cell depleting agent) so that immune recovery is faster after transplant is complete, but this may result in less effective transplant and/or a slower transplant procedure overall if a longer delay is required between initiation of lymphocyte depletion and the transplant.
  • conditioning regimens may use higher intensity lymphocyte depletion (e.g. higher / more frequent doses of cell depleting agent), but this may result in a longer time to immune recovery in the patient, and the depleting agent may remain in sufficient quantities to attack the transplanted cells.
  • a conditioning regimen including enzymatic inactivation of serum IgG in a subject results in significant benefits by making this balance easier to achieve, when used to rapidly inactivate an IgG-based agent used to reduce the numbers and/or down-modulate the activity of lymphocytes after it has had its positive effects, thereby reducing the time in which it may exert negative effects.
  • the present invention provides a conditioning regimen for the transplant of a cell to a subject, comprising:
  • the present invention also provides a method for the prevention or treatment of immune rejection of a cell transplant, the method comprising administering a cell transplant to a patient in accordance with the conditioning regimen of the invention.
  • the present invention also provides a method for the prevention or treatment of a disease or condition that is prevented or treated by cell transplant, the method comprising administering a cell transplant to a patient in accordance with the conditioning regime of the invention.
  • the disease or condition may be selected from Hematological malignancies; Solid tumor cancers; Hematologic diseases; Anemias; Myeloproliferative disorders; Metabolic disorders; Environmentally-induced diseases; Viral diseases; Autoimmune diseases; Lysosomal storage disorders; and Immunodeficiencies.
  • the present invention also provides a method for the treatment of alemtuzumab overdose, comprising administering to a patient in need thereof a therapeutically effective amount of an enzyme which inactivates serum IgG molecules in the subject.
  • polypeptide is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics.
  • polypeptide thus includes short peptide sequences and also longer polypeptides and proteins.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including both D or L optical isomers, and amino acid analogs and peptidomimetics.
  • patient and “subject” are used interchangeably and typically refer to a human.
  • References to IgG typically refer to human IgG unless otherwise stated.
  • Amino acid identity as discussed above may be calculated using any suitable algorithm.
  • the PILEUP and BLAST algorithms can be used to calculate identity or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300 ; Altschul, S, F et al (1990) J Mol Biol 215:403-10 .
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • HSPs high scoring sequence pair
  • T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787 .
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the UWGCG Package provides the BESTFIT program which can be used to calculate identity (for example used on its default settings) ( Devereux et al (1984) Nucleic Acids Research 12, 387-395 ).
  • the present invention provides a conditioning regimen for the transplant of a cell to a subject, comprising:
  • Step (a) may be conducted by any suitable method and using any suitable agent.
  • the same agent or combination of agents may be effective to reduce the numbers and/or down-modulate the activity of more than one type of lymphocyte. This may be referred to as lymphocyte depletion or lymphodepletion.
  • the agents may be referred to as lymphodepleting agents.
  • the agents are preferably administered at a dose sufficient to substantially reduce the numbers and/or substantially down-modulate the activity of lymphocytes in the subject.
  • Preferred agents target T lymphocytes and/or B lymphocytes, with T lymphocytes most preferred.
  • the agent may also deplete NK cells and/or non-malignant hematopoietic disease precursors.
  • lymphodepleting agents agents suitable for the depletion of lymphocytes (which may be referred to as lymphodepleting agents) are known in the art and include:
  • the conditioning regimen may additionally include administration also of a non-IgG based agent that is suitable to reduce the numbers and/or down-modulates the activity of lymphocytes in the subject.
  • a non-IgG based agent that is suitable to reduce the numbers and/or down-modulates the activity of lymphocytes in the subject.
  • a non-IgG based agent that is suitable to reduce the numbers and/or down-modulates the activity of lymphocytes in the subject.
  • a non-IgG based agent that is suitable to reduce the numbers and/or down-modulates the activity of lymphocytes in the subject.
  • a non-IgG based agent that is suitable to reduce the numbers and/or down-modulates the activity of lymphocytes in the subject.
  • any of busulfan, cyclophosphamide, fludarabine, treosulfan, cyclosporine, and tacrolimus may be used to deplete or inactive T cells
  • the present invention provides a conditioning regimen for the transplant of a cell to a subject, comprising:
  • the amount of said enzyme administered in step (b) is preferably sufficient to inactivate all or substantially all IgG molecules present in the serum of the subject. If necessary, more than one IgG-inactivating enzyme can be administered in combination, including simultaneously or sequentially, in any order.
  • serum IgG molecule(s) or "IgG molecule(s) present in the serum” refers to any gamma immunoglobulin (IgG1, IgG2, IgG3 and IgG4) molecule which is present in human tissue or in circulation prior to a method of the invention being carried out.
  • IgG molecules may have been produced endogenously from an individual's B-cells or may be exogenous gamma immunoglobulins which have been administered to a subject prior to the method of the invention being carried out - including any therapeutic IgG molecule of any origin.
  • Inactivation of serum IgG typically means a reduction in the Fc receptor interaction of IgG molecules.
  • Fc receptor refers to Fc gamma immunoglobulin receptors (FcyRs) which are present on cells.
  • FcyR refers to one, some, or all of the family of receptors comprising FcyRI (CD64), FcyRIIA (CD32A), Fc ⁇ RIIB (CD32B), FcyRIIC (CD32C), FcyRIIIA (CD16a) and Fc ⁇ RIIIB (CD16b).
  • FcyR includes naturally occurring polymorphisms of FcyRI (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIC (CD32C), FcyRIIIA (CD16a) and Fc ⁇ RIIIB (CD16b).
  • the enzyme used in the method of the invention may be any enzyme which inactivates serum IgG, but is typically an IgG cysteine protease which cleaves IgG such that the antigen binding domains and Fc interacting domains are separated from each other. In such cases, Fc receptor interaction of serum IgG molecules is reduced because the quantity of intact IgG molecules in the serum is reduced.
  • the enzyme may be an IgG endoglycosidase which cleaves a glycan structure on the Fc interacting domain of IgG, particularly the N-linked bi-antennary glycan at position Asn-297 (Kabat numbering). This glycan structure has a critical role in Fc receptor binding and complement activation.
  • the enzyme is preferably administered by intravenous infusion, but may be administered by any suitable route including, for example, intradermal, subcutaneous, percutaneous, intramuscular, intra-arterial, intraperitoneal, intraarticular, intraosseous or other appropriate administration routes.
  • the amount of the enzyme that is administered may be between 0.01 mg/kg BW and 2 mg/kg BW, between 0.01 mg/kg BW and 1 mg/kg BW, between 0.01 mg/kg BW and 0.5 mg/kg BW, between 0.01 and 0.3 mg/kg BW, preferably between 0.1 and 0.3 mg/kg BW, most preferably between 0.2 and 0.3 mg/kg BW.
  • Doses of approximately 0.12 mg/kg BW and approximately 0.24 mg/kg BW have been used in clinical settings.
  • a particularly preferred dose is approximately 0.25 mg/kg BW.
  • the enzyme may be administered on multiple occasions to the same subject, provided that the quantity of anti-drug antibody (ADA) in the serum of the subject which is capable of binding to the enzyme does not exceed a threshold determined by the clinician.
  • the amount of enzyme administered may be increased should a clinician consider this to be appropriate.
  • the quantity of ADA in the serum of the subject which is capable of binding to the protease may be determined by any suitable method, such as an agent specific CAP FEIA (ImmunoCAP) test or a titre assay. If ADA in the subject exceed said threshold, the condition regimen may include administration of an alternative enzyme.
  • the enzyme may be any of the following:
  • the IgG cysteine protease for use with the invention is specific for IgG.
  • the protease for use in the methods of the invention is IdeS ( I mmunoglobulin G-degrading enzyme of S. pyogenes), otherwise known as imlifidase.
  • IdeS is an extracellular cysteine protease produced by the human pathogen S. pyogenes. IdeS was originally isolated from a group A Streptococcus strain of serotype M1, but the ides gene has now been identified in all tested group A Streptococcus strains. IdeS has an extraordinarily high degree of substrate specificity, with its only identified substrate being IgG.
  • IdeS catalyses a single proteolytic cleavage in the lower hinge region of the heavy chains of all subclasses of human IgG. IdeS also catalyses an equivalent cleavage of the heavy chains of some subclasses of IgG in various animals. IdeS efficiently cleaves IgG to Fc and F(ab') 2 fragments via a two-stage mechanism. In the first stage, one (first) heavy chain of IgG is cleaved to generate a single cleaved IgG (scIgG) molecule with a non-covalently bound Fc/2 molecule. The scIgG molecule is effectively an intermediate product which retains the remaining (second) heavy chain of the original IgG molecule.
  • this second heavy chain is cleaved by IdeS to release a F(ab') 2 fragment and a homodimeric Fc fragment.
  • F(ab') 2 fragment may dissociate to two Fab' fragments and the homodimeric Fc may dissociate into its component monomers.
  • IdeS has been shown to be particularly effective at cleaving IgG in humans. The entire plasma IgG-pool is cleaved within minutes of dosing with IdeS, and IgG levels in blood remain low for more than a week until newly synthesized IgG appeared in plasma. This demonstrates that the entire extracellular IgG pool and not only the plasma pool (i.e. serum IgG molecules) is cleaved by IdeS ( Winstedt et al; PloS One 2015; 10(7): e0132011 ).
  • SEQ ID NO: 1 is the full sequence of IdeS including the N terminal methionine and signal sequence. It is also available as NCBI Reference sequence no. WP_010922160.1.
  • SEQ ID NO: 2 is the mature sequence of IdeS, lacking the N terminal methionine and signal sequence. It is also available as Genbank accession no. ADF13949.1.
  • the protease for use in the methods of the invention is IdeZ, which is a IgG cysteine protease produced by Streptococcus equi ssp. Zooepidemicus, a bacterium predominantly found in horses.
  • SEQ ID NO: 3 is the full sequence of IdeZ including N terminal methionine and signal sequence. It is also available as NCBI Reference sequence no. WP_014622780.1.
  • SEQ ID NO: 4 is the mature sequence of IdeZ, lacking the N terminal methionine and signal sequence.
  • the protease for use in the methods of the invention is a hybrid IdeS/Z, such as that of SEQ ID NO: 5.
  • the N terminus is based on IdeZ lacking the N terminal methionine and signal sequence.
  • the protease for use in the invention may comprise or consist of SEQ ID NO: 2, 4 or 5.
  • Proteases for use in the invention may comprise an additional methionine (M) residue at the N terminus and/or a tag at the C terminus to assist with expression in and isolation from standard bacterial expression systems.
  • Suitable tags include a histidine tag which may be joined directly to the C terminus of a polypeptide or joined indirectly by any suitable linker sequence, such as 3, 4 or 5 glycine residues.
  • the histidine tag typically consists of six histidine residues, although it can be longer than this, typically up to 7, 8, 9, 10 or 20 amino acids or shorter, for example 5, 4, 3, 2 or 1 amino acids.
  • the protease for use in the invention may comprise, consist essentially, or consist of the sequence of any one of SEQ ID NOs: 6 to 25. These sequences represent IdeS and IdeZ polypeptides with increased protease activity and/or reduced immunogenicity.
  • Each of SEQ ID NOs: 6 to 25 may optionally include an additional methionine at the N terminus and/or a histidine tag at the C terminus.
  • the histidine tag preferably consists of six histidine residues.
  • the histidine tag is preferably linked to the C terminus by a linker of 3x glycine or 5x glycine residues.
  • the protease for use in the invention may comprise, consist essentially, or consist of the sequence of any one of SEQ ID NOs: 56 to 69. These sequences represent IdeS polypeptides with increased protease activity and/or reduced immunogenicity.
  • Each of SEQ ID NOs: 56 to 69 may optionally include an additional methionine at the N terminus and/or a histidine tag at the C terminus.
  • the histidine tag preferably consists of six histidine residues.
  • the histidine tag is preferably linked to the C terminus by a linker of 3x glycine or 5x glycine residues.
  • the protease for use in the invention may comprise, consist essentially, or consist of the sequence of any one of SEQ ID NOs: 6 to 25, optionally with up to 3 (such as 1, 2 or 3) amino acid substitutions.
  • Each of SEQ ID NOs: 6 to 25 and variants thereof may optionally include an additional methionine at the N terminus and/or a histidine tag at the C terminus.
  • the protease for use in the invention may comprise, consist essentially, or consist of the sequence of any one of SEQ ID NOs: 56 to 69, optionally with up to 3 (such as 1, 2 or 3) amino acid substitutions.
  • Each of SEQ ID NOs: 56 to 69 and variants thereof may optionally include an additional methionine at the N terminus and/or a histidine tag at the C terminus.
  • the polypeptide of the invention is typically at least 100, 150, 200, 250, 260, 270, 280, 290, 300 or 310 amino acids in length.
  • the polypeptide of the invention is typically no larger than 400, 350, 340, 330, 320 or 315 amino acids in length. It will be appreciated that any of the above listed lower limits may be combined with any of the above listed upper limits to provide a range for the length the polypeptide of the invention.
  • the polypeptide may be 100 to 400 amino acids in length, or 250 to 350 amino acids in length.
  • the polypeptide is preferably 290 to 320 amino acids in length, most preferably 300 to 315 amino acids in length.
  • the primary structure (amino acid sequence) of a protease of the invention is based on the primary structure of IdeS, IdeZ or IdeS/Z, specifically the amino acid sequence of SEQ ID NO: 2, 4 or 5, respectively.
  • the sequence of a protease of the invention may comprise a variant of the amino acid sequence of SEQ ID NO: 2, 4 or 5, which is at least 80% identical to the amino acid sequence of SEQ ID NO: 2, 4 or 5.
  • the variant sequence may be at least 80%, at least, 85%, preferably at least 90%, at least 95%, at least 98% or at least 99% identical to the sequence of SEQ ID NO: 2, 4 or 5.
  • the variant may be identical to the sequence of SEQ ID NO: 2, 4 or 5 apart from the inclusion of one or more of the specific modifications identified in WO2016/128558 or WO2016/128559 .
  • Identity relative to the sequence of SEQ ID NO: 2, 4 or 5 can be measured over a region of at least 50, at least 100, at least 200, at least 300 or more contiguous amino acids of the sequence shown in SEQ ID NO: 2, 4 or 5, or more preferably over the full length of SEQ ID NO: 4 or 5.
  • the protease for use in the invention may be an IdeS, IdeZ or IdeS/Z polypeptide that comprises a variant of the amino acid sequence of SEQ ID NO:, 2, 4 or 5 in which modifications, such as amino acid additions, deletions or substitutions are made relative to the sequence of SEQ ID NO: 2, 4 or 5.
  • modifications are preferably conservative amino acid substitutions.
  • Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume.
  • the amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace.
  • the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid.
  • Conservative amino acid changes are well-known in the art.
  • IgG cysteine protease activity may be assessed by any suitable method, for example by incubating a polypeptide with a sample containing IgG and determining the presence of IgG cleavage products. Suitable methods are described in the WO2016/128559 . Suitable assays include an ELISA-based assay, such as that which is described in WO2016/128559 . In such an assay, the wells of an assay plate will typically be coated with an antibody target, such as bovine serum albumin (BSA). Samples of the polypeptide to be tested are then added to the wells, followed by samples of target-specific antibody that is antibody specific for BSA in this example.
  • BSA bovine serum albumin
  • the polypeptide and antibody are allowed to interact under conditions suitable for IgG cysteine protease activity. After a suitable interval, the assay plate will be washed and a detector antibody which specifically binds to the target-specific antibody will be added under conditions suitable for binding to the target-specific antibody.
  • the detector antibody will bind to any intact target-specific antibody that has bound to the target in each well. After washing, the amount of detector antibody present in a well will be proportional to the amount of target-specific antibody bound to that well.
  • the detector antibody may be conjugated directly or indirectly to a label or another reporter system (such as an enzyme), such that the amount of detector antibody remaining in each well can be determined.
  • At least one well on a given assay plate will include IdeS instead of a polypeptide to be tested, so that the potency of the tested polypeptides may be directly compared to the potency of IdeS. IdeZ and IdeS/Z may also be included for comparison.
  • assays may determine the potency of a tested polypeptide by directly visualizing and/or quantifying the fragments of IgG which result from cleavage of IgG by a tested polypeptide.
  • An assay of this type is also described in WO2016/128559 .
  • Such an assay will typically incubate a sample of IgG with a test polypeptide (or with one or more of IdeS, IdeZ and IdeS/Z as a control) at differing concentrations in a titration series. The products which result from incubation at each concentration are then separated using gel electrophoresis, for example by SDS-PAGE.
  • Whole IgG and the fragments which result from cleavage of IgG can then be identified by size and quantified by the intensity of staining with a suitable dye.
  • a polypeptide of the invention will typically produce detectable quantities of cleavage fragments at a lower concentration (a lower point in the titration series) than IdeZ and/or IdeS.
  • This type of assay may also enable the identification of test polypeptides that are more effective at cleaving the first or the second heavy chain of an IgG molecule, as the quantities of the different fragments resulting from each cleavage event may also be determined.
  • a polypeptide of the invention may be more effective at cleaving the first chain of an IgG molecule than the second, particularly when the IgG is an IgG2 isotype.
  • a polypeptide of the invention may be more effective at cleaving IgG1 than IgG2.
  • the enzyme may have IgG endoglycosidase activity, preferably cleaving the glycan moiety at Asn-297 (Kabat numbering) in the Fc region of IgG.
  • IgG endoglycosidase activity
  • EndoS Endo g l y cosidase of S. pyogenes
  • EndoS hydrolyzes the ⁇ -1,4-di- N- acetylchitobiose core of the asparagine-linked glycan of normally-glycosylated IgG.
  • the mature sequence of EndoS is provided as SEQ ID NO: 90.
  • the agent may be a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 90, or may be a homologue thereof from an alternative bacterium, such as Streptococcus equi or Streptococcus zooepidemicus, or Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica.
  • the agent may be CP40, EndoE, or EndoF 2 .
  • the agent may be a variant of the EndoS protein which comprises or consists of any amino acid sequence which has at least 80%, 85%, 90% or 95% identity with SEQ ID NO: 90 and has IgG endoglycosidase activity.
  • a variant of the EndoS protein may comprise or consist of an amino acid sequence in which up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more, amino acid substitutions, insertions or deletions have been made relative to the amino acid sequence of SEQ ID NO: 90, provided the variant has IgG endoglycosidase activity.
  • Said amino acid substitutions are preferably conservative. Conservative substitutions are as defined in the preceding section.
  • the agent may be a protein which comprises or consists of a fragment of SEQ ID NO: 90 and has IgG endoglycosidase activity, preferably wherein said fragment is 400 to 950, 500 to 950, 600 to 950, 700 to 950 or 800 to 950 amino acids in length.
  • a preferred fragment consists of amino acids 1 to 409 of SEQ ID NO: 90, which corresponds to the enzymatically active ⁇ -domain of EndoS generated by cleavage by the streptococcal cysteine proteinase SpeB.
  • the fragment may be created by the deletion of one or more amino acid residues of the amino acid sequence of SEQ ID NO: 90. Up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 550 residues may be deleted, or more. The deleted residues may be contiguous with other.
  • Any fragment or variant of SEQ ID NO: 90 preferably includes residues 191 to 199 of SEQ ID NO: 90, i.e. Leu-191, Asp-192, Gly-193, Leu-194, Asp-195, Val-196, Asp-197, Val-198 and Glu-199 of SEQ ID NO: 90.
  • These amino acids constitute a perfect chitinase family 18 active site, ending with glutamic acid.
  • the glutamic acid in the active site of chitinases is essential for enzymatic activity.
  • a variant of SEQ ID NO: 90 contains Glu-199 of SEQ ID NO: 90.
  • the variant of SEQ ID NO: 90 may contain residues 191 to 199 of SEQ ID NO: 90 having one or more conservative substitutions, provided that the variant contains Glu-199 of SEQ ID NO: 90.
  • the dose and timing of administration of a lymphodepleting agent comprising IgG molecules in accordance with step (a) of the method will typically be such that lymphocyte number and/or activity is substantially down-modulated in the individual subject.
  • Suitable dose schedules for known lymphodepleting agents are well known in the art.
  • the agent may be administered as one or more doses, administered spaced out over one or more days.
  • the first dose may be administered at any time, provided the final dose is administered at least one day prior to step (c).
  • the day prior to step (c) may be referred to as D -1, with the day of transplant referred to as D 0.
  • a typical schedule may have a first dose of agent taking place around 14, 13, 12, 11, 10, 8, 7, 6, 5, 4 or 3 days prior to step (c) (may be referred to as D -14, D -13, D -12 etc) followed by, for example, 2 to 7 (for a total of 3 to 8) or 2 to 5 (for a total of 3 to 6) separate, additional doses, typically one dose per day, provided that the final dose is administered at least one day prior to step (c), that is no later than D -1.
  • the IgG inactivating enzyme is administered in step (b) after the lymphodepleting agent in step (a), typically at a time point sufficiently after step (a) that the lymphodepleting agent has had the desired effects on lymphocyte numbers and/or activity, but sufficiently in advance of the subsequent cell transplant that inactivation by the enzyme will prevent negative effects that may be caused by the agent.
  • the timing of step (b) is typically at least 1 day after the conclusion of step (a), that is 1 day after the final dose of agent is administered in step (a).
  • Step (b) may take place on the same day as step (c), that is on D 0, but may preferably be at least 2, 3, 4, 5 or 6 hours prior to step (c).
  • the dose and timing of administration of the IgG inactivating enzyme will typically be sufficient to inactivate substantially all serum IgG molecules in the individual subject (i.e. including all of the lymphodepleting agent comprising IgG molecules), prior to administration of the transplant in step (c).
  • Suitable dose schedules for known IgG inactivating enzymes are well known in the art.
  • the inactivating enzyme should be administered at a time and dose such that the level of intact agent present in the subject immediately prior to transplant is reduced to less than 0. 1 ⁇ g/ml; where the lymphodepleting agent is ATG-G, the inactivating enzyme should preferably be administered at a time and dose such that the level of intact agent present in the subject immediately prior to transplant is reduced to less than 1 ⁇ g/ml; and where the lymphodepleting agent is Thymoglobulin, the inactivating enzyme should preferably be administered at a time and dose such that the level of intact agent present in the subject immediately prior to transplant is reduced to less than 0.3 ⁇ g/ml,
  • the conditioning regimen may additionally comprise one or more of:
  • Step (i) typically involves administering a dose of radiation which is sufficient to partially or totally eradicate (or ablate) the bone marrow of the subject. It is particularly applicable when the donor cells are HPSCs. Partial eradication by irradiation may be preferred since the side effects are typically less severe and also because it is desirable to retain some recipient bone marrow.
  • the ablation of recipient bone marrow creates space in the bone marrow for engraftment of donor cells, but also depletes lymphocytes in the subject and thus also reduces immune system activity in the same manner as step (a).
  • the conditioning regimen preferably includes at least (a), but most preferably includes at least (a) and (i).
  • step (a) it may be preferred in step (a) to use an irradiation free approach to depletion of subject HSPCs, such as administration of anti-CD 117 and/or anti-CD47.
  • an irradiation free approach to depletion of subject HSPCs such as administration of anti-CD 117 and/or anti-CD47.
  • This will create space for engraftment of donor HSPCs, but without some of the undesirable side-effects of irradiation.
  • the subject may also optionally receive an infusion of donor CD8-alpha cells, which may increase the frequency of stable chimerism in sensitized recipients.
  • Donor T cell infusion may promote donor HSPC engraftment by reducing survival of host T cells.
  • the present invention provides a method for the induction of hematopoietic chimerism in a subject, the method comprising conducting the conditioning regimen of the invention and subsequently administering HSPC to the subject in an amount sufficient and under conditions suitable to induce hematopoietic chimerism in the subject.
  • the method may alternatively be described as a method for the stable transplantation of HSPC.
  • the HSPC may be autologous (the patient's own cells are used) or syngeneic (the cells are from a genetically identical twin), or they may allogeneic (the cells come from a separate, non-identical donor).
  • Immune complications which reduce the likelihood of successful engraftment of HSPC in the recipient are most significant for allogeneic cells and thus the method of the invention is of greatest benefit with such cells.
  • immune complications can occur even with autologous cells if there is expression of a product to which the recipient has not previously been exposed. If an autologous cell has been genetically modified to express a gene therapy, the cell may be sufficiently altered to provoke an immune response. For example there may be an immune response to the expressed gene therapy product. Similar would apply if the HSPC has been genetically modified to express a different HLA type which is not matched to the HLA of the recipient.
  • the HSPC are preferably allogeneic, or are genetically modified autologous or syngeneic cells, for example Chimeric antigen receptor (CAR) T-cells.
  • the HSPC are most preferably allogeneic.
  • the HSPC are from a donor who is also the donor of another organ or tissue which is to be transplanted into the recipient. That is, the same donor provides both the HSPC and the other cell, organ or tissue.
  • HSPC are found in the bone marrow of adults, especially in the pelvis, femur, and sternum. They are also found in umbilical cord blood and, in small numbers, in peripheral blood. HSPC may be harvested from these locations using any suitable technique established in the art.
  • HSPC may be harvested from human bone marrow by aspirating directly from the centre of a bone of the donor with a large needle.
  • the posterior iliac crest is the usual site of harvest.
  • the technique is referred to as a bone marrow harvest and may be performed under local or general anesthesia.
  • the administration of HSPC may be described as a bone marrow transplant (BMT).
  • HSPC may be harvested from umbilical cord blood shortly after the birth of an infant.
  • the umbilical cord is double-clamped from the umbilicus and transacted between clamps.
  • the umbilical cord vein is then punctured under sterile conditions, and the blood flows freely by gravity into an anticoagulated sterile closed harvesting system, form which the HSPC may be isolated.
  • HSPC may be harvested from peripheral blood, typically by apheresis. However, because numbers of HSPC in peripheral blood are normally low, it is first necessary to mobilize HSPCs from the bone marrow. In a healthy donor, this can be achieved by administration of Granulocyte colony-stimulating factor (G-CSF). Alternative strategies may be required if the donor is not healthy. This may frequently be the case if the intended HSPC transplant is autologous.
  • G-CSF Granulocyte colony-stimulating factor
  • HSPC are preferably used as quickly as possible after harvesting (that is fresh), but may be cryopreserved for storage prior to thawing for use in the method of the invention.
  • Cryopreservation typically includes volume depletion by removal of red cells and plasma.
  • the quantity of stem cells in the harvest may be quantified, e.g. by flow cytometric analysis of a sample, to establish the proportion of cells which are positive for CD34 (a marker for stem cells).
  • the HSPC may be administered to the subject by any suitable method.
  • a preferred method is infusion, typically through a central line.
  • the patient may be kept in highly clean or sterile conditions, such as in a room with high-efficiency particulate air (HEPA) filters under positive pressure, before, during and after the infusion to reduce the risk of infection.
  • HEPA high-efficiency particulate air
  • the method may be monitored to determine that the HSPC transplant has successfully resulted in hematopoietic chimerism. This is achieved by determining the proportion of donor-derived hematopoietic cells present in a blood sample taken from the subject after a particular time interval, typically 28 days after administration of the HSPC.
  • hematopoietic chimerism may be defined as achieved if at least 5% of the lymphocytes and/or myeloid cells in the sample are found to be donor-derived, preferably if at least 5% of the lymphocytes in the sample are found to be donor-derived.
  • the chimerism is described as mixed if no more than 90% of the lymphocytes and/or myeloid cells in the sample are found to be donor-derived (that is at least 10% are still derived from the recipient), preferably if no more than 90% of the lymphocytes in the sample are found to be donor-derived (that is at least 10% of lymphocytes are still derived from the recipient).
  • the chimerism may be described as total if 98% or more of the lymphocytes and/or myeloid cells in the sample are found to be donor-derived.
  • Mixed chimerism is typically preferred for the methods of the invention, because the recipient will have a greater level of immunocompetence.
  • full chimerism may be beneficial in some circumstances, for example in the treatment of cancers such as leukemia where the goal is to eliminate host cells with the potential to cause cancer recurrence, replacing them with the transplanted HSPC.
  • the proportion of donor and recipient derived cells in a sample may be determined by any suitable method in the art, such as flow cytometric analysis as described in the Examples. Real-time PCR may also be used. Other methods are discussed in Agrawal et al Bone Marrow Transplantation 2004 (34) p-12.
  • the present invention provides a method for the prevention or treatment of a disease or condition in a subject.
  • the method comprises administering a cell transplant to the subject, wherein said administering comprises the conditioning regimen of the invention.
  • the invention also provides a method for the prevention or treatment of immune rejection of a cell transplant, the method comprising the conditioning regimen of the invention.
  • the cell transplanted may be of any type, including HSPC.
  • the HSPC may be a genetically modified HPSC, such that the method effectively comprises administering a gene therapy to the subject in which the HSPC is the vector.
  • the cell to be transplanted may originate from a different species to the recipient, that is it may be a xenotransplant. Suitable species for xenotransplantation into human recipients may include pigs or non-human primates. In such cases the HSPC may be genetically modified to aid with tolerance to the transplant.
  • the cell that is a xenotransplant may also be genetically modified.
  • the subject to be treated may be sensitized or highly sensitized.
  • sensitized it is meant that the subject has developed antibodies to human major histocompatibility (MHC) antigens (also referred to as human leukocyte antigens (HLA)).
  • MHC human major histocompatibility
  • HLA human leukocyte antigens
  • the anti-HLA antibodies originate from allogeneically sensitized B-cells and are usually present in patients that have previously been sensitized by blood transfusion, previous transplantation or pregnancy. Achieving hematopoietic chimerism in sensitized patients may reverse allosensitization, through the generation of specific tolerance in T and B cells, resulting in a reduction of donor specific immune responses such as DSA.
  • Whether or not a potential transplant recipient is sensitized may be determined by any suitable method.
  • a Panel Reactive Antibody (PRA) test may be used to determine if a recipient is sensitized.
  • a PRA score >30% is typically taken to mean that the patient is "high immunologic risk” or "sensitized”.
  • a cross match test may be conducted, in which a sample of the potential transplant donor's blood is mixed with that of the intended recipient.
  • a positive cross-match means that the recipient has antibodies which react to the donor sample, indicating that the recipient is sensitized and transplantation should not occur.
  • Cross-match tests are typically conducted as a final check immediately prior to transplantation.
  • the method may be for the prevention or treatment of any disease or condition that is treated by HSPC transplant.
  • Diseases or conditions typically treated by HSPC transplant may be acquired or congenital.
  • Acquired diseases or conditions that may be treated by HSPC transplant include:
  • Congenital diseases or conditions that may be treated HSPC transplant include:
  • the method of the invention may be for the prevention or treatment of the disease or condition to which said gene therapy is directed.
  • the invention also provides an enzyme which inactivates serum IgG molecules in a subject for use in a method for the prevention or treatment of a disease or condition, wherein the method is as described above.
  • the invention also provides the use of an enzyme which inactivates serum IgG molecules in a subject in the manufacture of a medicament, wherein the medicament is for the prevention or treatment of a disease or condition in a method as described above.
  • the enzymes used in the methods of the invention are polypeptides and may be produced by any suitable means.
  • a polypeptide may be synthesised directly using standard techniques known in the art, such as Fmoc solid phase chemistry, Boc solid phase chemistry or by solution phase peptide synthesis.
  • a polypeptide may be produced by transforming a cell, typically a bacterial cell, with a nucleic acid molecule or vector which encodes said polypeptide. Production of enzyme polypeptides by expression in bacterial host cells is described and exemplified in WO2016/128558 and WO2016/128559 .
  • compositions and formulations comprising polypeptides
  • the present invention also provides compositions comprising an enzyme for use in the methods of the invention.
  • the invention provides a composition comprising one or more polypeptides, and at least one pharmaceutically acceptable carrier or diluent.
  • the carrier(s) must be 'acceptable' in the sense of being compatible with the other ingredients of the composition and not deleterious to a subject to which the composition is administered.
  • carriers and the final composition are sterile and pyrogen free.
  • Formulation of a suitable composition can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the reasonably skilled artisan.
  • the enzyme can be combined with one or more pharmaceutically acceptable excipients or vehicles.
  • Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, reducing agents and the like, may be present in the excipient or vehicle.
  • Suitable reducing agents include cysteine, thioglycerol, thioredoxin, glutathione and the like.
  • Excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity.
  • compositions include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, thioglycerol and ethanol.
  • Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative.
  • Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (for e.g., a powder or granules) form for reconstitution with a suitable vehicle (e.
  • compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • a non-toxic parenterally-acceptable diluent or solvent such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.
  • compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems.
  • Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • the compositions may be suitable for administration by any suitable route including, for example, intradermal, subcutaneous, percutaneous, intramuscular, intra-arterial, intraperitoneal, intraarticular, intraosseous or other appropriate administration routes.
  • Preferred compositions are suitable for administration by intravenous infusion.
  • the invention also provides a kit for carrying out the methods described herein.
  • the kit of the invention may include an enzyme or a composition comprising an enzyme, as described above.
  • the kit may include means for administering the enzyme or composition to a subject.
  • the kit may include instructions for use of the various components in any method as described herein.
  • Example 1 optimal spacing of imlifidase and rATG
  • Imlifidase (conditionally authorised in the EU for kidney transplant desensitization) is a cysteine protease which cleaves all subclasses of human and rabbit IgG to a F(ab') 2 fragment and a dimeric Fc fragment.
  • Rabbit anti-thymocyte globulin (rATG) is a depleting antibody therapy approved for induction in kidney transplantation (it effects a large reduction in circulating T-lymphocytes).
  • Antibody-based therapies such as rATG may be inactivated if given with imlifidase. The purpose of this study was to investigate the cleavage of rATG by imlifidase.
  • rATG is rapidly cleaved and inactivated by imlifidase in human serum.
  • Example 2 use of imlifidase in preconditioning regimens with different antibody-based lymphodepleting agents
  • hematopoietic stem cell transplantation can be curative treatment options for patients with malignant diseases, e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and for patients with non-malignant diseases, e.g., immune deficiencies, Thalassemia and sickle cell disease.
  • rATG agents such as ATG-G or Thymoglobulin
  • anti-CD52 antibodies such as alemtuzumab are used prior to allogeneic T-cell depleted stem cell transplantation to allow a reduced intensity conditioning, leading to a significant reduction of graft versus host disease (GvHD) and engraftment failure.
  • Imlifidase is a cysteine protease derived from Streptococcus pyogenes which effectively hydrolyzes human and rabbit IgG, in the lower hinge region, into a F(ab')2 fragment and a dimeric Fc/2-fragment (2 ⁇ Fc/2).
  • the resulting F(ab')2 fragment retains full binding capacity to its antigens but the IgG-dependent Fc-dependent effector mechanisms, such as cell-mediated toxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are inactivated.
  • CDC cell-mediated toxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • imlifidase may be used as a tool to inactivate these drugs and eliminate toxicity, especially towards NK cells, before HSCT.
  • Fc-mediated, imlifidase may be used as a tool to inactivate these drugs and eliminate toxicity, especially towards NK cells, before HSCT.
  • imlifidase-inactivated high doses of ATG-G, Thymoglobulin and alemtuzumab leads to reduction of risk of GvHD, engraftment failure and relapse while boosting immune recovery by eliminating toxicity towards NK cells thus fostering a quicker immune recovery.
  • the suitability of imlifidase (or similar enzymes) in this context is demonstrated by the experiments performed here.
  • ATG-G, alemtuzumab, ATGAM, and thymoglobulin (1 mg/mL) were diluted in culture medium and incubated with 20 ⁇ g/mL imlifidase at 37°C for 2 hours.
  • the samples were diluted 10x in PBS and further diluted in 4X SDS sample buffer and incubated at 92°C for 3 min.
  • the samples were run on 4-20% Mini-PROTEAN ® tgx PRECAST SDS-PAGE gels (200V, 40 min), using 1 ⁇ Tris/Glycine/SDS-buffer.
  • 5 ⁇ L Precision Plus MW standard #161-0363 Bio-Rad was used.
  • the gels were activated for 2.5 min and exposed for 1 s using a Gel Doc EZ Imager system (Bio-Rad) and Image Lab 6.0 software (Bio-Rad).
  • rATG and alemtuzumab were hydrolyzed, using the FragIT Kit containing immobilized IdeS coupled to agarose beads on spin-columns, for 45 min at 37°C on a shaker.
  • the antibody fragments were collected after centrifugation and transferred to rabbit- or human-specific Fc-binding spin-columns to separate Fc- and F(ab') 2 -fragments.
  • the concentrations of the fragment fractions were determined by NanoDrop.
  • K562 is a target cell line from a CML patient in blast crisis carrying the BCR-ABL1 e14-a2 (b3-a2) fusion gene;
  • the feeder cell line K562-mbIL15-4-1BBL express membrane-anchored IL15. Both cell lines are grown in RPMI 1640 medium with FCS (10%), L-Glut (2 mM) and Pen/Strep (100 ⁇ g/ml).
  • eNKs The expanded NK cells (eNKs) were generated by incubating PBMCs from healthy donors with IL-2 (50 U/ml) in the presence of feeder cells (K562-mbIL15-4-1BBL) for 14 days. These cells can be frozen in aliquots for posterior use in additional experiments. Thawed eNKs were cultivated for 16h in RPMI-1640 medium supplemented with IL-2 (100 U/ml) and human AB serum (10%), then incubated with or without full length ATG-G or alemtuzumab or their respective F(ab') 2 - or 2 ⁇ Fc/2- fragments and human AB serum (25%) for 6h.
  • E effector
  • T calcein-labeled K562 wild type target cells
  • E:T calcein release assays for 2 hours on E:T, 10:1 proportion.
  • the amount of released calcein corresponds to the NK cell-mediated-cytotoxicity.
  • Concentrations ofF(ab') 2 - and 2 ⁇ Fc/2-fragments were chosen according to the percentage of molecular weight of full length alemtuzumab.
  • Peripheral blood was collected from a healthy volunteer in sodium heparin tube (BD-Plymouth). The whole blood was incubated at 37°C, 5% CO 2 for 4 and 16 hours with either alemtuzumab (Campath, anti-CD52, Genzyme) or Thymoglobulin (ATG, Genzyme), at final antibody concentrations of 100, 10, 1, 0.1 and 0 ⁇ g/mL. RBC were lysed upon sample incubation with Lysis Buffer (#349202, BD) for 5 minutes. The samples were centrifuged, and the supernatant discarded. The pellets were washed in FACS buffer and centrifuged at 1200 rpm for 3 min.
  • the pellets were resuspended in 50 ⁇ L PE-conjugated anti-CD3 antibody (Cat. No. MA1-10179, Thermo Scientific) and incubated at 4°C for 30 mins. FACS-buffer was added and the samples were analyzed on Accuri C6 flow cytometer.
  • Precision Plus MW standard 5 ⁇ L, was used as control.
  • the remaining of the samples were RBC-lysed and labelled with PE-conjugated anti-CD3 antibody (Cat. No. MA1-10179, Thermo Scientific) at 4°C for 30 mins and analyzed on Accuri C6 flow cytometer.
  • Alemtuzumab (Campath, anti-CD52, Genzyme), was pre-treated with different concentrations of imlifidase to generate F(ab')2, scIgG alemtuzumab. Samples were run on SDS-PAGE to confirm cleavage status.
  • Target NuDUL-1 cells were washed in PBS and stained with calcein at RT for 10 min in the dark. Cells were washed and resuspendded in R10 medium at a concentration of 1 ⁇ 10 6 cells/mL. THP-1 effector (E) cells, cultured in R10 medium, were washed and resuspended in PBS before staining with FarRed. After two washes, the cells were resuspended in R10 medium (1 ⁇ 10 6 cells/mL).
  • E effector
  • the NuDUL1 target cells were seeded in V-shaped wells (96-well plate), together with imlifidase generated F(ab')2, scIgG, or intact alemtuzumab at a final concentration of 10 ⁇ g/mL and incubated for 20 min prior to incubation with THP-1 effector cells. A sample without alemtuzumab was also prepared as negative control.
  • THP-1 effector cells 200 000/well
  • target cells 10 000/well
  • the plate was centrifuged and the supernatant discarded.
  • Resuspended cells were fixed with 4% PFA for 3 min before being resuspended in PBS (+0.5% BSA) after removal of the supernatant, and transfer to FACS tubes for analysis using the Accuri C6 flow cytometer.
  • the gels were activated for 45 s and scanned for 0.5-1 s using ChemiDoc Imaging system.
  • gel images from 15 individuals were evaluated using a visual scoring judging by the presence (+) or absence (-) of the F(ab')2 fragments over time.
  • Autologous or allogeneic cell therapies can be curative treatment options for patients with malignant (e.g. ALL, AML) and non-malignant diseases (e.g. immune deficiencies, Thalassemia).
  • Safe and efficacious treatments need to overcome several hurdles, including the preconditioning of the host to eliminate malignant or defective hematogenic cells and to allow for the establishment of donor BMC.
  • One of the challenges is to find treatment regimens that strikes a balance between sufficiently conditioning the host by eliminating pathogenic cells without compromising a swift reestablishment of the hematopoietic system by donor BMC. If this balance is not achieved, complications such as GvHD, graft failure (GF) and life-threatening infections may occur.
  • GvHD graft failure
  • GF graft failure
  • irradiation and lymphodepleting drugs like ATG or alemtuzumab.
  • the IgG-based biologics rabbit ATG and anti-CD52 alemtuzumab bind and kill potentially both, donor- and host lymphocytes.
  • using high doses of these drugs leads to a desired reduction of graft-versus-host disease (GvHD), but at the cost of delayed immune reconstitution or even increased host engraftment failure, thereby enhancing the risk of severe infectious complications and overall survival (OS).
  • GvHD graft-versus-host disease
  • Persisting levels of alemtuzumab or of ATG in the patient's blood (and bound to endothelial cells) creates yet another level of intricacy, delaying the immune recovery after HSCT which are associated with an increased risk of severe viral reactivations.
  • Fig. 3 shows that polyclonal rabbit ATG (ATG-G or Thymoglobulin), as well as, the human IgG1-based mAb alemtuzumab are efficiently cleaved in vitro by imlifidase, into their respective 2 ⁇ Fc/2 and F(ab')2 fragments by imlifidase in buffer.
  • Rabbit ATG and human IgG1-based alemtuzumab are thus examples of biologics that can be uncoupled from their Fc-effector functions and potentially be inactivated in vivo through imlifidase cleavage.
  • the horse polyclonal IgG thymoglobulin cannot be cleaved by imlifidase.
  • NK cells were incubated with different concentrations of intact antibody (IgG) or their respective in vitro -cleaved 2 ⁇ Fc/2 and F(ab')2 fragments.
  • rATG-F(ab')2 The cytotoxic potency of rATG-F(ab')2 towards eNK cells was reduced compared to the equivalent concentrations of intact IgG ( Fig.4 ). After incubation of NK cells with 100 ⁇ g/mL intact rATG only 20 % of the K562 target cells were lysed, demonstrating an impairment of the NK cells. In contrast, the equivalent dose of rATG F(ab')2 still allowed the NK cells to lyse most of their target tumor cells. This thus demonstrates that rATG F(ab')2 is less cytotoxic than intact rATG, even if it retains some of its cytotoxic activity at very high doses.
  • the protective effect of imlifidase cleavage of biologics is even more prominent when using the anti-CD52 targeting antibody alemtuzumab.
  • a concentration of 2 ⁇ g/mL alemtuzumab efficiently inactivates the NK-effector cells, exhibiting an equivalent reduction of K562 target cell lysis at 100 ⁇ g/mL alemtuzumab.
  • equivalent fragment doses of 100 ⁇ g/mL alemtuzumab, i.e. 75,3 ⁇ g/mL F(ab')2 or 24,7 ⁇ g/mL 2 ⁇ Fc/2 fragments have no negative effect on NK cells.
  • Neither 2 ⁇ Fc/2 purified from imlifidase-cleaved rATG ( Fig. 4c ) nor from alemtuzumab had a blocking effects of NK cell activity.
  • ADCP is one of the effector mechanisms by which alemtuzumab can mediate the removal of CD52-positive target cells.
  • Alemtuzumab was incubated with different concentrations of imlifidase to address the question which activity the different degrees of IgG digestion have on ADCP.
  • the digests were run on SDS-PAGE to facilitate the visualization of the samples that were fully cleaved by imlifidase into F(ab')2 and 2 ⁇ Fc/2 fragments, predominantly into scIgG, or intact alemtuzumab as positive control ( Fig.5a ).
  • NuDUL1 target cells were incubated with either intact IgG, scIgG or fully imlifidase-cleaved F(ab')2 and 2 ⁇ Fc/2 alemtuzumab fragments to test their ability to mediate ADCP by monocytic THP1 cells.
  • a dose titration of the different alemtuzumab preparations shows that scIgG alemtuzumab completely loses its ability to opsonize CD52-positive NuDUL1 target cells at around 2 ⁇ g/mL, while intact alemtuzumab displays the same potency at 2 ⁇ g/mL and 30 ⁇ g/mL.
  • Completely cleaved alemtuzumab into 2 ⁇ Fc/2 and F(ab')2 by imlifidase fully loses its ability to mediate ADCP, even at 30 ⁇ g/mL.
  • Flow cytometry shows that imlifidase at 1.9 ⁇ g/mL is able to cleave endogenous IgG ( Fig.7a ) and able to block the cytotoxic effect of 100 ⁇ g/mL alemtuzumab on CD3-positive T-cells ( Fig.7b ).
  • the horse IgG-based drug ATGAM is not cleavable by imlifidase ( Fig. 3d ), and its cytotoxic activity in whole blood is not reduced even at 30 ⁇ g/mL imlifidase ( Fig. 8b ), while the endogenous IgG pool is fully cleaved ( Fig.8a ).
  • CDC The role of CDC in the early cytotoxic activity of alemtuzumab (30 ⁇ g/mL) and horse ATGAM (100 ⁇ g/mL) was shown by pre-incubating whole blood with the C5 inhibitor eculizumab.
  • Single- and fully-cleaved alemtuzumab by imlifidase show the same degree of CDC blocking ( Fig.9 ).
  • Imlifidase has not only the ability to cleave serum IgG in vivo but it also to cleaves therapeutic antibodies such as rabbit ATG and alemtuzumab into 2 ⁇ Fc/2 and F(ab')2 fragments in the presence of serum matrix.
  • the detachment of the Fc from IgG allows the F(ab')2 fragment not only to be eliminated faster due to its reduced molecular size but also due to the lack of FcRn-recycling, otherwise observed with intact IgG.
  • hematopoietic stem cell transplantation can be curative treatment options for patients with malignant diseases, e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and for patients with non-malignant hematological diseases, e.g., immune deficiencies, Thalassemia and sickle cell disease.
  • Preconditioning of the host is not only needed to create space for the donor HSC in the hematological niches, but also to suppress graft rejection by host immune cells, to avoid GvHD, and to eliminate the pathogenic host cells.
  • Regimens including total body irradiation and administration of e.g. IgG-based biologics need to be fine-tuned to balance the desired with the negative effect from those treatments.
  • the resulting F(ab')2 fragment retains binding capacity to antigens (e.g. CD52) but their Fc-dependent effector mechanisms, such as complement-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are eliminated. Since the mechanisms of action of rATG, Thymoglobulin and alemtuzumab are, to a large extent, Fc-mediated, imlifidase can be used as a tool to inactivate these drugs and limit cytotoxicity towards NK cells before HSCT.
  • antigens e.g. CD52
  • Fc-dependent effector mechanisms such as complement-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are eliminated. Since the mechanisms of action of rATG, Thymoglobulin and alemtuzumab are, to a large extent, Fc-mediated
  • imlifidase is also of use in cases where some of the cytotoxic effector mechanism is mediated through the crosslinking of receptors, which would be achieved due to the accelerated elimination rate of F(ab')2 fragments compared to intact IgG.
  • IgG removal like plasmapheresis or plasma exchange, are inferior, since the drug antibodies i.e. rATG or alemtuzumab cannot be removed completely from the patient due to inevitable backflow from the tissue and interstitial fluids (Achini et al., 2020; Zhang et al., 2019). The same is true for already cell-bound antibodies. Imlifidase on the other hand, will inactivate Fc functions right away in the blood compartment as well as in interstitial fluid and even of already cell bound antibodies.
  • imlifidase might also be useful in cases where no antidote is available for an accidental overdose of IgG-based drugs (van der Zwan et al., 2018), which need to be inactivated instantaneously. Imlifidase is also superior because it can be administered within minutes, whereas plasmapheresis or plasma exchange are more strenuous for patients.
  • Example 3 dosing and timing intervals for imlifidase with alemtuzumab, ATG-G and Thymoglobulin

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